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Bacillus subtilis M406 and application thereof to preparation of bacteriocin and cellulase

A technology of Bacillus subtilis and cellulase, which is applied in the field of microorganisms, can solve problems such as unfavorable fermentation, reduce oxygen dissolution in the medium, and achieve the effects of preventing drug resistance and improving nutritional value.

Active Publication Date: 2019-01-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Inoculating Bacillus subtilis into it and standing it for fermentation will often form a layer of bacteria film on the surface of the medium, which will reduce the dissolution of oxygen in the medium. During the suspension culture process, bacteria will settle at the bottom, which is not conducive to the progress of fermentation.

Method used

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  • Bacillus subtilis M406 and application thereof to preparation of bacteriocin and cellulase
  • Bacillus subtilis M406 and application thereof to preparation of bacteriocin and cellulase
  • Bacillus subtilis M406 and application thereof to preparation of bacteriocin and cellulase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Isolation and identification of Bacillus subtilis M406 strain

[0073] 1. Isolation of Bacillus subtilis M406 strain

[0074] 1.1 Separation

[0075] In a high-quality pig farm of Huazhong Agricultural University, we took fresh feces of adult commercial pigs and Meishan pigs and placed them in sterile tubes; diluted pig feces with sterile PBS, and took 10 -3 ~10 -6 4 dilutions of 100μl each of the bacteria solution, spread on the primary screening medium plate, inverted culture at 37℃ for 1d ( figure 1 ). Among them, the primary screening medium: CMC-Na 10g, Na 2 HPO 4 2.5g, KH 2 PO 4 1.5g, peptone 1g, yeast powder 0.5g, agar 15g, distilled water to make the volume to 1L.

[0076] 1.2 Initial screening:

[0077] Select well-growing single colonies and plant them on the primary screening medium plate (the numbers of single colonies isolated from commercial pigs start with "S", and the numbers of single colonies isolated from Meishan pig start with "M"), and inverted ...

Embodiment 2

[0088] 1. Identification of the antibacterial effect of Bacillus subtilis M406

[0089] Bacillus subtilis (Bacillus subtilis) M406 was inoculated into LB medium according to the inoculation amount of 1%, and cultured at 37°C with 200r / min shaking for 18 hours. The culture solution was centrifuged at 12000r / min for two minutes, and the supernatant was passed through a 0.22μm filter. Then use the punch method to determine the diameter of the inhibition zone. Three repeated tests were done for each strain.

[0090] The test found that Bacillus subtilis (Bacillus subtilis) M406 broth had antibacterial effect on G+ bacteria S.aureus, S.suis, G-bacteria E.coli and S.typhimurium. S.aureus and S.suis are common bacteria that cause infection, while E.coli and S.typhimurium are important causes of diarrhea in piglets.

[0091] This shows that Bacillus subtilis (Bacillus subtilis) M406 broth can inhibit the growth of E. coli and S. typhimurium.

[0092] 2. Determination of the nature of the an...

Embodiment 3

[0100] 1. Preparation of bacteriocins

[0101] The method for preparing bacteriocin using Bacillus subtilis M406 includes the following steps:

[0102] 1) Strain activation: Bacillus subtilis (Bacillus subtilis) M406 strain was grafted on the LA solid medium and cultured at a temperature of 30~37℃ for 12~24h to obtain a single colony; among them,

[0103] The concentration composition of LA solid medium is: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15g / L;

[0104] 2) Seed solution preparation: Inoculate a single colony in LB liquid medium, culture it for 5-24 hours at a temperature of 30-37°C and a shaker speed of 150-220r / min until the mid-logarithmic growth of the strain. Obtain seed liquid; wherein, the LB liquid medium formula: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L;

[0105] 3) Liquid shake flask fermentation: Put the seed liquid into the sterilized bacteriocin-producing medium, culture and ferment for 12-24h at a temperature of 30~37℃ and a shaker speed of 150~...

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Abstract

The invention discloses Bacillus subtilis M406 and an application thereof to preparation of bacteriocin and cellulase. The Bacillus subtilis M406 strain is preserved with the number of CCTCC NO:M 2018234. One probiotic strain, namely, the Bacillus subtilis M406 is screened out on the basis of the thought of taking one character as main and various characters as auxiliaries, and use value of probiotic is increased. Antibacterial active substances which can be produced by the Bacillus subtilis M406 can inhibit various harmful bacteria including S. typhimurium, Salmonella enteritidis, S.aureus, E.coli and Listeria monocytogenes in animal intestines. The probiotic can ferment multiple saccharides for acid production, and pH of intestinal juice is reduced. The Bacillus subtilis M406 produces cellulase, so that structural carbohydrates in coarse fodder can be degraded effectively, part of NSP can be transformed into soluble saccharides, fats, proteins and amino acids, and nutritional value of the coarse fodder is increased.

Description

Technical field [0001] The present invention relates to the technical field of microorganisms, in particular to a Bacillus subtilis M406 and its application in the preparation of bacteriocins and cellulase. Background technique [0002] Bacillus, G + Bacteria, 0.7-0.8μm×1.0-1.5μm, are straight or nearly straight rod-shaped cells, mostly moving, flagella laterally growing, strictly aerobic or facultative anaerobic. Heterotrophs, aerobic metabolism, fermentation metabolism or both, some can reduce nitrate, and most produce catalase. When in an adverse environment, the spores of Bacillus are endophytic, and there is not more than one spore in a cyst, and the spore is the dormant state of Bacillus. The spores are resistant to stress, and can withstand temperature, dryness, acid and alkali, disinfectants and many unfavorable factors. The spores will re-convert into vegetative cells in a suitable environment for growth and reproduction. Some bacillus can produce a variety of digesti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P21/00C12N9/42A61K38/16A61P31/04C12R1/125
CPCA61K38/164A61P31/04C12N9/2437C12P21/00C12N1/205C12R2001/125Y02A50/30
Inventor 晏向华王新锴胡军侯奇良牛耀嵘
Owner HUAZHONG AGRI UNIV
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